Kei Maruyama

Expression of necdin, an embryonal carcinoma-derived nuclear protein, in developing mouse brain

Necdin is a polypeptide sequence encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells. We have examined the expression of necdin and its mRNA in cultured cells and mouse brain by Northern blot analysis and immunohistochemistry. Among various established cell lines including neuroblastoma and glioma cells, only differentiated embryonal carcinoma cells (P19 and F9) expressed necdin mRNA. Necdin immunoreactivity was localized in the nuclei of differentiated neurons derived from P19 cells. Necdin mRNA was detected throughout brain regions of adult mouse; the relative abundances in the hypothalamus and midbrain were the highest, whereas those in the olfactory bulb and cerebellum were the lowest. In developing mouse brain, necdin mRNA was expressed during early periods of neuronal generation and differentiation, and the peak levels were attained during postnatal days 1-4. Necdin immunoreactivity was not detected in the neural stem cells on embryonic day 10, but was concentrated in the nuclei of brain cells, mostly neurons, at advanced stages of differentiation. The majority of differentiated neurons in the brain had necdin-immunoreactive nuclei on postnatal day 33. Thus, necdin may represent a valuable molecular marker for differentiated neurons both in vitro and in vivo.

Molecular Dissection of Domains in Mutant Presenilin 2 That Mediate Overproduction of Amyloidogenic Forms of Amyloid β Peptides INABILITY OF TRUNCATED FORMS OF PS2 WITH FAMILIAL ALZHEIMER’S DISEASE MUTATION TO INCREASE SECRETION OF Aβ42

Mutations in presenilin (PS) 1 or PS2 genes account for the majority of early-onset familial Alzheimer’s disease, and these mutations have been shown to increase production of species of amyloid β peptide (Aβ) ending at residue 42, i.e.the most amyloidogenic form of Aβ. To gain insight into the molecular mechanisms whereby mutant PS induces overproduction of Aβ42, we constructed cDNAs encoding mutant and/or truncated forms of PS2 and examined the secretion of Aβ42 from COS or neuro2a cells transfected with these genes. Cells expressing full-length PS2 harboring both N141I and M239V mutations in the same polypeptide induced overproduction of Aβ42, although the levels of Aβ42 were comparable with those in cells engineered to express PS2 with one or the other of these PS2 mutations. In contrast, cells engineered to express partially truncated PS2 (eliminating the COOH-terminal third of PS2 while retaining the endoproteolytic NH2-terminal fragment) and harboring a N141I mutation, as well as cells expressing COOH-terminal fragments of PS2, did not overproduce Aβ42, and the levels of Aβ42 were comparable with those in cells that expressed full-length, wild-type PS2 or fragments thereof. These data indicate that: (i) the Aβ42-promoting effects of mutant PS2 proteins reach the maximum level with a given single amino acid substitution (i.e. N141I or M239V); and (ii) the expression of full-length mutant PS2 is required for the overproduction of Aβ42. Hence, cooperative interactions of NH2- and COOH-terminal fragments generated from full-length mutant PS2 may be important for the overproduction of Aβ42 that may underlie familial Alzheimer’s disease.

Amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum

The amino acid sequence of vitamin D‐dependent calcium‐binding protein from bovine cerebellum has been determined. It is composed of 260 amino acid residues and its N‐terminus is acetylated. The molecular mass is calculated to be 29 851 Da. The presence of six calcium‐binding sites (I–VI) has been proposed, two of them (sites II and VI) have lost their calcium‐binding function through amino acid replacements, and the other four are able to bind calcium. six calcium‐binding domains are supposed to be derived from two gene duplications of the two ancestral calcium‐binding domains. In comparison with the sequence of chick intestinal calcium‐binding protein deduced from a cDNA sequence [(1985) Nucleic Acids Res. 13, 8867‐8881], the bovine calcium‐binding protein is two amino acid residues shorter at the N‐terminus and the other parts show 78.5% identity.

Purification of vitamin D-dependent 28,000-Mr calcium-binding protein from bovine cerebellum and kidney by calcium-dependent elution from DEAE-cellulose DE-52 column chromatography

A vitamin D-dependent calcium-binding protein of 28,000 Mr was purified by a convenient method from the high-ammonium sulfate-saturated fraction (70 to 100% saturation) of bovine cerebellum and kidney. This method is based on the calcium-dependent DEAE-cellulose column chromatography that reflected the specific feature of this protein: different eluting salt concentrations in the presence or absence of Ca2+. The procedure of purification was easily performed by the detection of calcium-binding protein using 45Ca autoradiography. The purified calcium-binding protein showed the same molecular weight and calcium-dependent change of mobility in nondenaturing gel electrophoresis as vitamin D-dependent calcium-binding protein. The 28,000-Mr calcium-binding protein was induced in kidney by the injection of vitamin D in vitamin D-deficient rats.

A new species of enkephalin precursor mRNA with a distinct 5′-untranslated region in haploid germ cells

To elucidate the primary structure of preproenkephalin (A) mRNA expressed by haploid germ cells (round spermatids) in rat testis, we have screened a λgt11 cDNA library for preproenkephalin cDNA inserts. The largest cDNA insert contained a protein‐coding sequence encoding 269 amino acid residues as well as 327 and 309 bases of the 5′‐ and 3′‐untranslated regions, respectively. The protein‐coding region plus 3′‐untranslated region of the mRNA was over 99% homologous to that of brain preproenkephalin mRNA, whereas the 5′‐untranslated region contained a distinct sequence including a partial sequence of intron A of the preproenkephalin gene [(1984) J. Biol. Chem. 259, 14301–14308; (1984) J. Biol. Chem. 259, 14309–14313]. Northern blot analysis using a 5′‐end‐specific probe showed that this type of preproenkephalin mRNA exists exclusively in the germ cells.

Association of chicken pectoralis muscle phosphorylase with the Z-line and the M-line of myofibrils: Comparison with ‘amorphin’, the amorphous component of the Z-line

By immunofluorescence technique, glycogen phosphorylase, one of the soluble glycolytic enzymes, was shown to be localized in the Z-line of chicken pectoralis muscle myofibrils, in addition to the M-line, as previously reported by Heizmann, C.W. and Eppenberger, H.M. (Heizmann, C.W. and Eppenberger, H.M. (1978) J. Biol. Chem. 253, 270-277). After extraction of thick filaments by a solution containing pyrophosphate and high salt (Hasselbach-Schneider solution), or after extraction of thin and thick filaments by a solution containing 0.6 M KI, phosphorylase still remained in the Z-line. Amorphin (Mr 85 000) was reported by Chowrashi, P.K. and Pepe, F.A. (Chowrashi, P.K. and Pepe, F.A. (1982) J. Cell Biol. 94, 565-573) as a new Z-line amorphous component. The amino acid composition of amorphin reported by them was very similar to that of phosphorylase b reported by Heizmann and Eppenberger. The partially purified 85 kDa protein, according to Chowrashi and Pepe, showed cross-reactivity to anti-phosphorylase serum and phosphorylase activity. Although amorphin was reported to be eluted from DEAE-column chromatography around the gradient of 0.5 M KCl, little protein was eluted around 0.5 M KCl in our experiments, and the 85 kDa protein which we identified as phosphorylase b was eluted around 0.2 M KCl. Hence, it should be said that the major 85 kDa protein extracted according to Chowrashi and Pepe was phosphorylase b.